Alzheimer's disease (AD), an age-associated dementing disorder, currently affects 4 million Americans and is the fourth leading cause of death in the US. The prevalence of AD doubles every 5.1 years from 1% of Americans ages 65-69 to ˜39% of Americans age 90-95 (Jorm et al. Acta Psychiat Scand 76:465-79 (1987). With aging of the “baby boom” generation, the largest segment of the US population, there may be 14 million Americans with AD by 2040 unless preventative strategies are developed. With a mean survival time of 8.1 years and a range of 1 to 25 years AD is a growing socio-economic and medical problem with an estimated cost of 100 billion dollars per year in the US alone.
Risk factors associated with AD include aging, presence of APOE-4 alleles, family history, head injury, low educational attainment and low linguistic ability early in life. Although mutations in presenilin 1 and 2 and the amyloid precursor protein (APP) are associated with familial AD (FAD) these cases account for less than 5% of all AD subjects. Currently, there are no known genetic mutations associated with sporadic AD.
Pathologically, the AD brain is characterized by a loss of total brain weight, neuron loss and shrinkage, synapse loss, neuropil thread formation, abundance of neurofibrillary tangles (NFT) and senile plaques (SP) and by a proliferation of reactive microglia and astrocytes in particular brain regions including the hippocampus, amygdala and nucleus basalis of Meynert (nbM).
The two hallmark pathologic markers of AD examined at autopsy are NFT and SP. NFT are intracellular deposits of hyperphosphorylated tau, a microtubule associated protein in neuronal axons. The normal function of tau is to bind tubulin in axonal microtubules and promote stabilization (Goedert et al. EMBO J 8:393-99 (1989), Buee et al. Brain Res Brain Res Rev 33:95-130 (2000)). As such, tau is essential for axonal function and transport. Tau has 30 potential phosphorylation sites (Iqbal et al. J Neural Transm Suppl 62:309-319 (2002); Buee et al. Brain Res Brain Res Rev 33:95-130 (2000) and abnormal phosphorylation is observed in AD NFT leading to a loss of ability to bind microtubules (Iqbal et al. J Neurol 59:213-222 (2000).
Senile plaques are extracellular deposits composed of a central core of amyloid β peptide (Aβ), a 40 or 42 amino acid polypeptide derived from the amyloid precursor protein (APP). Surrounding the amyloid core are dystrophic neurites and reactive glia. Formation of Aβ occurs through combined action of beta and gamma secretase cleavage of APP leading to the neurotoxic Aβ40/42. Mutations in APP associated with FAD are associated with increased generation of Aβ40/42. Mutations in presenilin-1 and 2 (gamma secretases) are also associated with FAD and lead to increased production of Aβ40/42. Although NFT and SP are the hallmark neuropathologic markers of AD, it is unclear if they are involved in the etiology of AD or are merely end products of neurodegeneration. Additionally, it has been suggested that AD pathology may begin 20 to 30 years before the onset of clinical symptoms (Davies et al. Neurology 38:1688-93 (1988); Price and Morris Ann Neurol 45:358-68 (1999)). During the preclinical period, NFT and SP increase until reaching a critical threshold when clinical symptoms appear.
Without a clear genetic influence on sporadic AD several etiologic/pathogenic hypotheses have been suggested including energy metabolism defects, deficiencies of neurotrophic factors, glutamate toxicity, mitochondrial defects, trace element toxicity and free radical mediated toxicity.
Clinically, AD is characterized by a loss of spontaneity, drive and initiative, a progressive worsening of memory, behavioral changes, onset between 40 and 90, and the absence of systemic disorders or other brain diseases. The main clinical feature of AD is a progressive cognitive decline leading to memory loss. The memory dysfunction involves impairment of learning new information which is often characterized as short-term memory loss. In the early (mild) and moderate stages of the illness, recall of remote well-learned material may appear to be preserved, but new information cannot be adequately incorporated into memory. Disorientation to time is closely related to memory disturbance.
Language impairments are also a prominent part of AD. These are often manifest first as word finding difficulty in spontaneous speech. The language of the AD patient is often vague, lacking in specifics and may have increased automatic phrases and cliché´s. Difficulty in naming everyday objects is often prominent. Complex deficits in visual function are present in many AD patients, as are other focal cognitive deficits such as apraxia, acalculia and left-right disorientation. Impairments of judgment and problem solving are frequently seen.
Non-cognitive or behavioral symptoms are also common in AD and may account for an even larger proportion of caregiver burden or stress than the cognitive dysfunction. Personality changes are commonly reported and range from progressive passivity to marked agitation. Patients may exhibit changes such as decreased expressions of affection. Depressive symptoms are present in up to 40% of AD subjects. A similar rate for anxiety has also been reported. Psychosis occurs in 25% of AD subjects. In some cases, personality changes may predate cognitive abnormality.
Currently, clinical diagnosis of AD is based on structured interviews (patient histories), and neuropsychological examinations coupled with imaging or neurophysiological scans (CT, MRI, PET and/or SPECT scans and EEG) to rule out other explanations of memory loss including temporary (depression or vitamin B12 deficiency) or permanent conditions (stroke) and is based on NINCDS-ADRDA Work group criteria (McKhann et al. Neurology 34:939-48 (1984) and the American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (4th Ed. Washington D.C., Am Psychiatric Assoc. (1997).
Unfortunately, clinical diagnostic methods are not foolproof. Evidence based review of current literature shows clinical diagnostic accuracy of 65 to 90%. Higher accuracy rates are generally associated with specialized centers (memory disorder clinics) focused on memory disorders whereas lower rates are likely associated with primary care physicians. Additionally, accuracy of the clinical diagnosis is likely lower during early stages of the disease when symptoms are difficult to differentiate from normal age-associated cognitive decline. More recently, studies suggest that a condition termed mild cognitive impairment (MCI) represents prodromal AD and if diagnosed early represents the best opportunity for pharmaceutical intervention. The clinical criteria used for diagnosis of MCI are those of Petersen et al. (Arch Neurol 56:303-308 (1999) and include: 1) memory complaints corroborated by an informant, 2) objective memory impairment for age and education, 3) normal general cognitive function, 4) intact activities of daily living, and 5) the subject does not meet criteria for dementia.
Further complicating diagnosis and treatment of AD is the lack of a reliable biomarker that specifically identifies AD subjects, particularly early in the prodromal stage of the disease (MCI).
In view of the magnitude of the public health problem posed by AD, considerable research efforts have been undertaken to elucidate the etiology of AD as well as to identify biomarkers, characteristic proteins or metabolites objectively measured as an indicator of pathogenic processes, that can be used to diagnose and/or predict whether a person is likely to develop AD.
Most studies of biomarkers of AD have focused on measurement in the cerebrospinal fluid (CSF). CSF is produced in the choroid plexus, a leaf like structure that projects into the lateral, third and fourth ventricles of the brain (Huhmer et al. Disease Markers 22:3-26 (2006)) and is in direct contact with the extracellular space of the brain. Because of its intimate contact with the brain, pathogenic changes in the brain that result in alterations in proteins/peptides would likely be reflected in the CSF.
A number of U.S. patents and published applications relate to methods for diagnosing AD, including U.S. Pat. Nos. 4,728,605, 5,874,312, 6,027,896, 6,114,133, 6,130,048, 6,210,895, 6,358,681, 6,451,547, 6,461,831, 6,465,195, 6,475,161, 6,495,335, 2005/0244890, and 2005/0221348. Additionally, a number of reports in the scientific literature relate to certain biochemical markers and their correlation/association with AD, including Fahnestock et al., 2002, J. Neural. Transm. Suppl. 2002(62):241-52; Masliah et al., 1195, Neurobiol. Aging 16(4):549-56; Power et al., 2001, Dement. Geriatr. Cogn. Disord. 12(2):167-70; and Burbach et al., 2004, J. Neurosci. 24(10):2421-30. Additionally, Li et al. (2002, Neuroscience 113(3):607-15) and Sanna et al. (2003, J. Clin. Invest. 111(2):241-50) have investigated Leptin in relation to memory and multiple sclerosis, respectively.
Three different biomarkers in CSF have been particularly well documented: neuronal thread protein, tau (total; T-tau and various phosphorylated forms; P-tau) and derivatives of amyloid precursor protein (APP) including Aβ40 and Aβ42.
Neuronal thread protein is described to be overexpressed in brain neurons in AD patients. A quantitative test for measuring levels of a specific type of neuronal thread protein (AD7c-NTP) in CSF and urine has been developed. Quite a number of studies have evaluated CSF-tau as an ante-mortem marker for AD mainly using enzyme-linked immunoabsorbent assays (ELISA) as the measurement assay. In past studies, total tau (T-tau) has been measured although there is an increasing body of literature also describing the analysis of phosphorylated (P-tau) variants of the same protein involved in the formation of NFTs. ELISAs that can distinguish between the major form of Aβ ending at amino acid 40 (Aβ40) and the senile plaque forming species ending at position 42 (Aβ42) have also been developed and evaluated extensively for CSF analysis. These three assays, either used individually, or in the case of tau and Aβ 42, in combination, have not demonstrated the required sensitivity and specificity values for routine clinical use, particularly for early diagnosis and discrimination between AD and other non-AD dementias. In addition, attempts to measure tau and Aβ42 in blood have been met with limited success, further restricting their widespread adoption into clinical practice.
A wide spectrum of other aberrations, other than NTP, Tau and Aβ, has been reported in AD patient CSF. Many of the identified (protein sequence confirmed) CSF markers reported herein have been shown to be either increased or decreased in AD patients versus normal individuals. For example, the protein Ubiquitin is known to complex with hyperphosphorylated Tau during maturation of NFTs in the brains of AD patients (Iqbal et. al. J Neural Transm Suppl. 53:169-80 (1998)). Ubiquitin levels in CSF of AD and neurological control groups have been shown to be significantly higher than those of non-neurological aged controls (Wang et. al. Acta Neuropathol (Berl). 82(1):6-12 (1991); Kudo et. al. Brain Res. 639(1):1-7 (1994)).
The acute phase/inflammatory protein alpha(1)-antichymotrypsin (ACT) is overproduced in the AD brain. ACT also can promote the formation of, and is associated with, neurotoxic amyloid deposits (Potter et. al. Neurobiol Aging. 22(6):923-30 (2001)). The levels of ACT in both serum and CSF are significantly and specifically higher in patients with Alzheimer-type dementia than in control subjects (Matsubara et. al. Ann Neurol. 28(4):561-7(1990)). There is a particularly close association of increases in CSF-ACT with late onset AD (Harigaya et. al. Intern Med. 34(6):481-4 (1995)).
Chromogranin A (CrA) is the major protein of large dense-core synaptic vesicles and may be of value as a biochemical marker for synaptic function in AD. One report described no difference between AD, vascular dementia, and age-matched control groups except when comparing a familial subtype (AD Type I) with controls where there was a statistically significant elevation of CSF CrA in the diseased individuals (Blennow et. al. Dementia. 6(6):306-11 (1995)).
Beta-2-Microglobulin (β2M) is an initiator of inflammatory responses modulated by interferons and certain cytokines (Hoekman et.al. Neth. J. Med. 28:551-557 (1985)). A proteome analysis of CSF by two-dimensional electrophoresis (2D-gel) has shown a significant increase of β2M in AD patients (Davidsson et al., Neuroreport, 13:611-615 (2002)), and more recently these results were confirmed by SELDI analysis (Carrette, O. et. al., Proteomics, 3:1486-1494 (2003)).
Transthyretin (TTR) has been shown to interact with Aβ, possibly preventing amyloid formation in biological fluids and in the brain. (Tsuzuki et al., Neurosci Lett, 10:171-174 (2000)). One identified TTR isoform was shown to be increased in AD-CSF using 2D gel analysis of a small number of AD and control patients (Davidsson, supra.). However, this result conflicts with other reports showing a clear decrease of TTR in CSF from AD patients compared with controls (Serot et. al. J Neurol Neurosurg Psychiatry. 63(4):506-8 (1997); Riisoen et. al. Acta Neurol Scand. 78(6):455-9 (1998)). This decrease is also negatively correlated with the senile plaque (SP) abundance (Merched et. al. FEBS Lett. 425(2):225-8 (1998)).
Cystatin C, a cysteine protease inhibitor, has been implicated in the neurodegenerative and repair processes of the nervous system, and the deposition of the same protein together with beta amyloid peptide was found as cerebral amyloid angiopathy (CAA) in different types of dementias (Levy et.al. J. Neuropathol. Exp. Neurol. 60:94-104). Full length Cystatin C was found as a CSF marker for AD in a previous SELDI profiling study (Carrette, supra.). A relative blood-brain barrier (BBB) dysfunction is associated with AD among very elderly individuals. The CSF/serum albumin ratio can be used as a measure of BBB function. Mean CSF/serum albumin ratio has been reported to be higher in all dementias studied, including AD, than in nondemented individuals (Skoog et al, Neurology. 50:966-71 (1998)).
Transferrin (TF) plays a role in anti-oxidant defense in serum and is also produced in the brain where its role in oxidative stress is unclear. A study on Down's syndrome patients suffering from progressive dementia showed decreased levels of TF when compared to age-matched controls with no neurological disease (Elovaara Acta Neurol Scand. 69(5):302-5(1994)).
Prostaglandin-D-Synthase (PDS) functions to convert prostaglandin H2 to prostaglandin D2 and has been identified in several studies of CSF (Harrington et al. Appl Theoret Electrophor 3:229-34 (1993); Hiraoka et al. J Chormatogr A 802:143-48 (1998); Kiraoka et al. Electorphorsis 22:3433-3437 (2001); Kawashima Mod. Pathol 14:197-201 (2001); Mase et al. Neurosci Lett 270:188-190; Mase Neurosci Res 47:455-459 (2003); Melegos et al. Prostaglandins 54:463-474 (1997)). Additionally, PDS demonstrates altered isoforms in neurologic disorders including AD and Parkinson's disease.
The present inventors have addressed the above-identified needs by identifying a protein-protein complex that is present in AD and MCI CSF and not age-matched control subjects. Additionally, the present inventors have invented a diagnostic test based on the detection of the protein-protein complex that identifies AD and MCI subjects with a high degree of specificity. Levels of these protein-protein complexes can serve as biomarkers to preferentially identify subjects with AD from age-matched control subjects or subjects with other neurologic disorders. In addition, these biomarker complexes can be used to identify subjects with mild cognitive impairment (MCI), the earliest clinical manifestation of AD.
The diagnostic test of the present invention is of considerable interest and benefit because of its ability to identify subjects with AD, particularly early in the progression of the disease (MCI). The tests of the present invention may also be used to monitor efficacy of treatment.